Process for improving the flavor of foods by the addition of 5&#39;-nucleotides



United States Patent 3,104,171 PROCESS FOR lMPRDVlNG THE FLAVGR 6F FOGDSBY THE ADDITIUN OF '-NUCLEOTEDES Kiuichiro Sakaguchi, Tokyo, andMasajiro Kihi and Akira Kunina'ka, Choshishi, Chibaken, Japan, assignorsto Yamasa Shoyu KllfrflSliliil Keisha (doing business as Yamasa Sh'oyuCo., Ltd), Choshi, Japan, a Japanese corporation No Drawing. Originalapplication Aug. 22, 1958, Ser. No. 756,541. Divided and thisapplication Dec. 8, 1961, Ser. No. 158,1(98

5 Claims. (ill. 99-140) This invention relates to a process forproducing the solution containing 5-nucleotides(adenosine-S'unonophosphate, guanosine 5' Inonophosphate, uridine-S'-monophosphate, cytidine-S'-monophosphate, inosine-5- monophosphate,xanthosine 5 monophosphate) from ribonucleic acid by microbial5'-phosphodiesterase action, and to an application of the 5-nucleot-idesas special seasonings. The object of this invention is to produceflavorous 5-nucleotides, which were so tar prepared generally only byorganic synthesis or by extraction from tissues of various organismssuch as mammalian muscle, economically and in good yield fromribonucleic acid, using the enzymes of microorganisms.

This is a division of copending application Serial No. 756,541, filedAugust 22, 1958.

Chemical degradation of ribonucleic acid results in formation of 3- and2'-nucleotides and does not result in formation of 5-nucleotides.Furthermore general ribonucleodepolynrerases, without distinction of thekind of origins, degrade ribonucleic acid into 3 (or 2)- nucleotides butnot into 5-nucleotides. Only so-calied unspecific phosphodiesterasesfrom snake venom or in testinal mucosa degrade ribonucleic acid into5'-nucleotides. However, it is very diflicult to obtain a large amountof these enzymes. 5'-nucleotides can be produced by means of organicsynthesis but said process is very troublesome and not economical too.Thus, hitherto, the production of 5'-nucleotides was very difficult, andespecially economical mass production thereof was quite impossible.

We have found that some strains of bacteria, yeasts, and molds contain 5'-phosphodiesterases which specifical- 1y hydrolyze the5'-phosphodiester linkages in ribonucleic acid and produce four5'-nucleotides: adenosine-S-monophosphate, guanosine 5 monophosphate,cytidine-S-monophosphate, and uridine-5-monophosphate. Especially theseveral strains which belong to the following genuses have beenrecognized to contain strong 5'-phosphodiesterase: Bacillus,Streptomyces, Torula, Zygosaccharomyces, Penicillium and Aspergillus.Thus the basis of the production of 5- nucleotides by microorganismsaccording to the present invention has been established for the firsttime.

This invention has been accomplished on the basis of the aboveconfirmation. Therefore, the present invention provides a process forthe production of 5'-nucleotides which is characterized in this thatribonucleic acid is degraded into Snucleotides by 5-phophodiesterasewhich is contained in living cells, dry cells, culture filtrates or cellextracts of microorganisms described above. The microorganismscontaining 5'-phosphodiesterase are able to be grown on either solidmedia or liquid media. For economical mass production, however, liquidmedia are more appropriate. As the components of the culture medium, theconventional carbon and nitrogen sources and several inorganic salts maybe employed eifectively.

3,104,171 Patented Sept. 17, 1963 This invention includes both one stepmethod and two step method. In one step method, both growing ofmicroorganism and enzymic degradation of ribonucleic acid are carriedout simultaneously, employing culture medium containing ribonucleicacid. In two step method, growing of microorganism and enzyrnicdegradation of ribonucleic acid are carried out separately.

According to the present invention, it is not necessary to purifyribonucleic acid before its enzymic degradation. Crude solutioncontaining ribonucleic acid, such as yeast extracts, may be used as anappropriate starting material. Furthermore microbial cells cultivatedfor producing 5'- phosphodiesterase are eliectively utilized too as asource of ribonucleic acid.

Free 5-nucl'eotides or their alkali salts obtained by the processing asdescribed above enhance or increase the flavor of the foods, beverages,and seasonings in which they are placed. This flavoring action is causedby the synergy between 5-nucleotides and amino acids or organic acid-s.According to our discovery, purine and pyrimidine bases, theirnucleosides, and their 2- and 3- nucleotides have little flavor, while5-nucleotides, especially inosine-5'-monophosphate,guanosine-Y-monophosphate, and xanthosine-S-monophosphate, have veryagreeable good taste. Furthermore, there is specific synergy in tastebetween 5'-nucleotides and amino acids or organic acids. Among variousamino acids, glut'arnic and aspartic acids were recognized to beespecially elfective in the synergy with 5-nucleotides. General foods,beverages, and seasonings contain considerable quantity of arnino acidsor organic acids as main flavoring components, but scarcely contain5-nucleotides. Therefore, it seems that the role of 5-nucleotides inflavoring is Very important. For example, the good taste of soups ormeat extracts, containing small quantity of 5'-nucleotides, may beperhaps caused mainly by the synergy between 5'-nucleotides and aminoacids.

This invention relates also to the application of 5- nucleotides basedon the utilization of aforesaid synergy between 5'-nucleoti-des andamino acids or organic acids. The application of 5'-nucleotidesaccording to the present invention comprises adding one or more of5'-nucleotides to general foods or beverages such as meat products,soups, roux, vinegar, various dressings, sauces, curry powder andvarious drinks including wine, to counteract the displeased pungencywhich spoils the taste qualities of foods or beverages, and to enhanceor increase the flavor specifically according to the synergy between 5'-nucleotides and amino acids or organic acids present in the foods orbeverages. In case of application for the foods or beverages containingno amino acids, it is more effective to add monosodium glutamic acid and5'-nucleotides together. 5-nucleotides may be also employed to enrichspecifically the seasonings containing amino acids. The bittersubstances in the crude preparations of 5- nucleotides can be readilyremoved by cation exchange resin. Both crude and purified preparationsof 5'-nucleotides are useful.

' According to the present invention, alkali salts of 5'- nucleotidesmay be also employed similarly as free 5- nucleotides since there is nosignificant diiference between their flavoring action.

The invention is illustrated but not limited by the following examples.

Example 1 phate, 0.04% of magnesium sulfate, and 0.04% of calciumchloride were sterilized and inoculated with a pure culture ofPenicillium citrinum. After surface culture at 3 30 C. for five days themycelial deck was separated from the culture broth, and washed withsterilized water. The washed mycelial deck was incubated with 50 ml. of0.5% yeast ribonucleic acid solution containing 0.01 N sodium fluorideat 30 C. After 22.5 hours the deck was removed. The resulting reactionmixture was recognized to contain 70-80 mg. of mononucleotides, 80-90mg. of nucleosides, and 70-80 mg. of undepolyrnerized polynucleotides.The mononucleotides, which are contained in above mixture, wereidentified as cytidine-'-monophosphate, adenosine-5'-monophosphate,inosine-5'-monophosphate, uridine-S-rnonophosphate, andguanosine-5'-mono phosphate. The identification was carried out asfollows: 23 ml. of .the reaction mixture were adjusted to pH 8.5 withstrong sodium hydroxide solution. 2.5 ml. of 20% barium acetate solutionwere added thereto. The precipitate of barium phosphate formed wasremoved. The supernatant was adjusted to pH 5.0 with a small quantity ofacetic acid. 1 ml. of mercuric acetate solution (20% in 2% acetic acid)was added. The precipitate was centrifuged, washed and suspended inwater. Into the suspension hydrogen sulfide gas was introduced toseparate nucleotides. The mixture was filtered and the precipitate waswashed with hot water. The solution resulting from the washing wascombined with the supernatant and a portion of the combined solutionswas adjusted to pH 8.5, and charged into anion exchange resinDowex-l-Cl- X-4 (200-400 mesh) column with a diameter of 1.0 cm. and 23cm. in height, and was eluted with 0.003 N (No. l-No. 216 test tubes)and 0.010 N hydrogen chloride (No. 217-No. 302 test tubes). Each 80drops of the eluate were collected into a test tube and optical densityat 260 mg of each eluate was read. Five ultraviolet absor'oing fractionsA, B, C, D and E were obtained. The

4% growth medium was shaken on a reciprocating shaker at 30 C. After 7days culture filtrates were concentrated in vacuo, and then dialyzedagainst running Water overnight. To the dialyzed solution 4 volumes ofethanol were added. The resulting precipitate, which was rich in5-phosphodiesterase activity, was dried up in a desiccator and wasemployed as an enzyme preparation. From 1 litre of culture filtratesabout 1 g. of the prepanation was obtained. 1 g. of this preparation wasincubated with 200 ml. of 5% ribonucleic acid at 65 C. and pH 5.0. Underthese conditions phosphomonoesterase and adenyl deanrin'ase were almostinactive, while 5-phosphodiesterase was recognized to be very active.

The reaction proceeds as described below:

Incubation time, min n 0' 10 30' 60} 90 5-nucleutirles formation fromribonucleic acid, percent 68 06 07 Inorg. 1. formation from5-nuclcoti1les,

percent 0 3. 0 3. 5 4. 5 5. 7

Example 3 100-500 mg. of the crude mixture of adenosine-5'-monophosphate, guanosine-S-monophosphate, cytidinepropenues of thesefractions are tabulated as follows: 5'-monophosphate, andurid1ne-5-monophosphate ob- Nueleotidc fraction obtained Standardsubstance A B C D E Cyti- Adcno- Adeno- Inosine- Urldinc-Guanodinesinesine- 5-mono- 3-monosine- Mixture 013- No of test tubes3-mono- 3-mono- 5-monophosphos- 3-mouonuclcotidcs phosphosphosphatepliatc phosphate phatc phato pirate 21-24 43-54 121-122 221-237 261-281Amax (my) 275 258 .249 262 257 278 257 257 253 Percent color, 7 min 78.782.2 81. 0 39. G Development, 15 mln. 94.2 98.9 97.2 72.8 (Pentose-), 25min 100.0 100.0 100.0 04. 7 (Orcinol), 35 min-.. 96.0 06. 8 96.3 99. 0(Reaction), 45 min. 03. 9 92. 6 92.8 100.0 Carbazolo reaction Blue BlueBlue Purple Blue Purple Distance migrated from origin to anode side byelectrophoresis, cm 4.9 4.9 12.5 14.7 7.7 4.9 5.0 4.5 14 8,8. 7,4 7NaIOm'osaniline reaction.

1 Ultraviolet absorption spectra of standard substances were measured in0.1 N H01. 1 The technique employed was essentially the same as thatdescribed by Albaum and Umbreit (J. Biol. Chem, 167, 360, (1047)). 8Starting line was at 5 cm. from the end on the cathode side, and 26 cm.from the end on the anode side.

For the formation of the strong 5-phosphodiesterase, shaking culture ismore eiiective than surface culture at least in case of the strainemployed in Example 1.

The culture medium employed in Exarnple 1 was inoculated withPenicillium citrinum. The inoculated tained in Example 2 were added to 1litre of sauce. (Practically, 2-10 ml. of reaction mixture were employedagainst 1 litre of sauce.) By this treatment displeased pungency ofsauce was counteracted perfectly, and the good flavor was greatlyincreased. Thus the sauce became fiavorous almost beyond recognition.The mixture of deaminated 5-nucleotides containinginosine-5'-monophosphate, xanthosine-5-monophosphate, and uridine-5-monophosphate, was also employed to improve the taste of sauce. Themixture of 5'-nucleotides was able to be kept in a form of dry powderfor a long time.

Example 4 Monosodium glutamate was coated with purifiedinosine-5'monophosphate disodium salt or guanosine-5'- monophosphoricacid. The ratio of monosodium glutamate to inosine-S'-monophosphate orguanosine-5'-monophosphate was 5-15:1. The resultant superior seasoningwas recognized to have remarkable flavoring properties for all kind ofdishes.

Example T o 120 g. of soup potage powder (corresponding to 1800 ml. offin'al volume) 1 to 2 g. of purified inosine-5'- monophosphaite disodiumsalt or guanosine-5-monophosphoric acid were added. From the resultantenriched powder, remarkable fiavorous soup was prepared. Instead of thepurified nucleotide preparations each crude preparation or mixture of5-n-uoleotides was also employed satisfactorily.

In case of soup consom-me being prepared inos=ine5- monophosphate,guanosine-S-rnonophosphate, and mixture of 5-nucleotides were employedeffectively too.

What is claimed is:

1. A process for improving and increasing specifically the flavor offoods, beverages, and seasonings containing amino acids which comprisesadding thereto at least one compound selected from the group consistingof free 5'- nucleotides and their alkali salts.

2. A process for improving and increasing specifically the flavor offoods, beverages, and seasonings containing substantially no amino acidswhich comprises adding thereto at least one 5'-nucleotide in addition tomono sodium glutamate.

3. A process for preparing 5 -nucleotides which comprises cultivatingthe microorganism selected from the group consisting of Bacillus,Streptomyces, Torula, Zygosaccharomyces, Aspergillus and Penicilliumwhich contain 5-phosphodiesterase, degrading ribonucleic acid to5'-nucleotides by 5'phosphodiestenase which is contained in the culturedmaterial selected from the group consisting of living cells, dry cells,culture filtrates and cell extracts resulting fronr the cultivation ofsaid microorganism, and adding said 5'-nucleotides to a materialselected from the class consisting of foods, beverages and seasoningscontaining amino acids.

4. A process for preparing 5-nucleotides which comprises cultivating themicroorganism selected from the group consisting of Bacillus,Streptomyces, Torula, Zygosaccharomyces, Aspergillus and Penicilliumwhich contain 5-phosphodiesterase, degrading ribonucleic acid to5'-nucleotides by 5'-phosphodiesterase which is contained in thecultured material selected from the group consisting of living cells,dry cells, culture filtrates and cell extracts resulting from thecultivation of said microorganism, adding an alkali salt to a solutioncontaining said 5'-nucleotides and admixing the solution with a materialselected from the class consisting of foods, beverages and seasoningscontaining amino acids.

5. A process for preparing 5-nucleotides Which comprises cultivating themicroorganism selected from the group consisting of Bacillus,Streptomyces, T orula, Zygosaccharomyces, Aspergillus and Penicilliumwhich contain 5'-phosphodiesterase, degrading ribonucleic acid toReferences Cited in the file of this patent Cohn et al.: Journal ofBiological Chemistry, vol. 203, July-August 1953, pages 319 to 331.

Dixon et al.: Enzymes, published by Academic Press Inc., New York, 1958,pages 190, 191 and 690.

1. A PROCESS FOR IMPROVING AND INCREASING SPECIFICALLY THE FLAVOR OFFOODS, BEVERAGES, AND SEASONINGS CONTAINING AMINO ACIDS WHICH COMPRISESADDING THERETO AT LEAST ONE COMPOUND SELECTED FROM THE GROUP CONSISTINGOF FREE 5''NUCLEOTIDES AND THEIR ALKALI SALTS.